M971 chimeric antigen receptors

ABSTRACT

The invention provides a chimeric antigen receptor (CAR) comprising an antigen binding domain comprising SEQ ID NOs: 1-6, a transmembrane domain, and an intracellular T cell signaling domain. Nucleic acids, recombinant expression vectors, host cells, populations of cells, antibodies, or antigen binding portions thereof, and pharmaceutical compositions relating to the CARs are disclosed. Methods of detecting the presence of cancer in a mammal and methods of treating or preventing cancer in a mammal are also disclosed.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application No.61/717,960, filed on Oct. 24, 2012, the entire contents of which areincorporated herein by reference.

INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

Incorporated by reference in its entirety herein is a computer-readablenucleotide/amino acid sequence listing submitted concurrently herewithand identified as follows: one 31,786 Byte ASCII (Text) file named“714144_ST25.txt,” dated Aug. 30, 2013.

BACKGROUND OF THE INVENTION

Cancer is a public health concern. Despite advances in treatments suchas chemotherapy, the prognosis for many cancers, including hematologicalmalignancies, may be poor. For example, it has been estimated that morethan 45,000 deaths were expected from non-Hodgkin's lymphoma andleukemia in the United States in 2000 (Greenlee et al., CA Cancer J.Clin., 50:7-33 (2000)). Accordingly, there exists an unmet need foradditional treatments for cancer, particularly hematologicalmalignancies.

BRIEF SUMMARY OF THE INVENTION

An embodiment of the invention provides a chimeric antigen receptor(CAR) comprising an antigen binding domain comprising SEQ ID NOs: 1-6, atransmembrane domain, and an intracellular T cell signaling domain

Further embodiments of the invention provide related nucleic acids,recombinant expression vectors, host cells, populations of cells,antibodies, or antigen binding portions thereof, and pharmaceuticalcompositions relating to the CARs of the invention.

Additional embodiments of the invention provide methods of detecting thepresence of cancer in a mammal and methods of treating or preventingcancer in a mammal.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)

FIGS. 1A-1G are graphs showing % lysis of target ⁵¹Cr labeled leukemiacell lines REH (A), SEM (B), NALM6-GL (C), KOPN8 (D), Daudi (E), Raji(F), and K562 (G) by effector human T cells that were untransduced(mock, •; circles) or transduced with the following CARs: HA22-SH-secondgeneration, version 1 (▾), m971-second generation, version 1 (▴; opentriangle), or CD19-specific CAR (▪; squares) at various effector totarget ratio (E:T) ratios.

FIGS. 2A-2C are graphs showing percent lysis of target leukemia celllines Raji (A), NALM6-GL (B), or K562 (C) by effector untransduced Tcells (mock, •; circles), m971-second generation, version 1 CAR (▴), orm971-third generation CAR (▾) at various E:T ratios.

FIGS. 3A-3C are graphs showing the amounts of interferon (IFN)-γ (pg/ml)secreted by T cells that were untransduced (mock) or transduced with oneof the following CARs: anti-CD19, m971-second generation version 1 (SEQID NO: 23), m971-third generation (SEQ ID NO: 24), HASH22-secondgeneration version 1, HASH22-second generation version 2, orHASH22-third generation upon co-culture with leukemia cell linesNALM6-GL (CD22^(low)) (A), Raji (CD22^(hi)) (B), or K562 (CD22-negative)(C).

FIGS. 3D-3F are graphs showing the amounts of interleukin (IL)-2 (pg/ml)secreted by T cells that were untransduced (mock) or transduced with oneof the following CARs: anti-CD19, m971-second generation version 1 (SEQID NO: 23), m971-third generation (SEQ ID NO: 24), HASH22-secondgeneration version 1, HASH22-second generation version 2, orHASH22-third generation upon co-culture with leukemia cell linesNALM6-GL (CD22^(low)) (A), Raji (CD22^(hi)) (B), or K562 (CD22-negative)(C).

FIGS. 3G-3I are graphs showing the amounts of tumor necrosis factor(TNF)-α (pg/ml) secreted by T cells that were untransduced (mock) ortransduced with one of the following CARs: anti-CD19, m971-secondgeneration version 1 (SEQ ID NO: 23), m971-third generation (SEQ ID NO:24), HASH22-second generation version 1, HASH22-second generationversion 2, or HASH22-third generation upon co-culture with leukemia celllines NALM6-GL (CD22^(low)) (A), Raji (CD22^(hi)) (B), or K562(CD22-negative) (C).

FIG. 4A is a graph showing bioluminescent signals (photons/s/cm²/sr)generated by the reaction of luciferase (transfected into leukemiacells, which were injected into mice) with luciferin that was injectedinto the mice, measured over a time period of 30 days. The mice weretreated with control T cells (“mock,” untransduced, •; circles) or Tcells transduced with HASH22 CAR-second generation, version 1 (▪;squares) or m971-second generation version 1 (triangles, SEQ ID NO: 23).Higher photons/s/cm²/sr values indicates greater tumor burden.

FIG. 4B is a graph showing percent survival of mice treated with controlT cells (“mock,” untransduced, squares) or T cells transduced withHASH22 CAR-second generation version 1 (circles) or 971 CAR-secondgeneration version 1 (triangles, SEQ ID NO: 23) over 30 days. (Mock v.HA22, p=0.0019; mock v. m971, p=0.0019; m971 v. HA22, p=0.003).

FIG. 5 is a table showing bioluminescent images of mice having leukemiathree, five, and eight days after treatment with untransduced T cells(mock) or 15×10⁶, 5×10⁶, 1×10⁶, or 1×10⁵ T cells that were transducedwith a nucleotide sequence encoding a m971-second generation, version 2CAR (SEQ ID NO: 22). A change in shading from grey to white indicatesincreased tumor burden.

FIG. 6 is a graph showing the total number of photons emitted in thebioluminescence of the mice shown in FIG. 5 over a period of 44 days.The total number of photons was quantified and plotted, and the averagesand standard deviations are shown for each time point. Mice were treatedwith untransduced T cells (mock) (plus sign) or 15×10⁶ (diamond), 5×10⁶(flower), 1×10⁶ (cross), or 1×10⁵ (asterisk) T cells that weretransduced with a nucleotide sequence encoding a m971-second generation,version 2 CAR (SEQ ID NO: 22).

DETAILED DESCRIPTION OF THE INVENTION

An embodiment of the invention provides a chimeric antigen receptor(CAR) comprising an antigen binding domain comprising SEQ ID NOs: 1-6, atransmembrane domain, and an intracellular T cell signaling domain.

A CAR is an artificially constructed hybrid protein or polypeptidecontaining the antigen binding domains of an antibody (e.g., singlechain variable fragment (scFv)) linked to T-cell signaling domains.Characteristics of CARs include their ability to redirect T-cellspecificity and reactivity toward a selected target in anon-MHC-restricted manner, exploiting the antigen-binding properties ofmonoclonal antibodies. The non-MHC-restricted antigen recognition givesT cells expressing CARs the ability to recognize antigen independent ofantigen processing, thus bypassing a major mechanism of tumor escape.Moreover, when expressed in T-cells, CARs advantageously do not dimerizewith endogenous T cell receptor (TCR) alpha and beta chains.

The phrases “have antigen specificity” and “elicit antigen-specificresponse” as used herein means that the CAR can specifically bind to andimmunologically recognize an antigen, such that binding of the CAR tothe antigen elicits an immune response.

The CARs of the invention have antigen specificity for CD22. CD22 is alineage-restricted B cell antigen belonging to the immunoglobulin (Ig)superfamily. CD22 is expressed in 60-70% of B cell lymphomas andleukemias (e.g., B-chronic lymphocytic leukemia, hairy cell leukemia,acute lymphocytic leukemia (ALL), and Burkitt's lymphoma) and is notpresent on the cell surface in early stages of B cell development or onstem cells. Vaickus et al., Crit. Rev. Oncol./Hematol., 11:267-297(1991); Bang et al., Clin. Cancer Res., 11: 1545-50 (2005).

Without being bound to a particular theory or mechanism, it is believedthat by eliciting an antigen-specific response against CD22, theinventive CARs provide for one or more of any of the following:targeting and destroying CD22-expressing cancer cells, reducing oreliminating cancer cells, facilitating infiltration of immune cells totumor site(s), and enhancing/extending anti-cancer responses. BecauseCD22 is not expressed in early stages of B cell development or on stemcells, it is contemplated that the inventive CARs advantageouslysubstantially avoid targeting/destroying stem cells and/or B cells inearly development stages.

An embodiment of the invention provides a CAR comprising an antigenbinding domain of the m971 antibody (“m971”). The antigen binding domainof m971 specifically binds to CD22. In this regard, a preferredembodiment of the invention provides CARs comprising an antigen-bindingdomain comprising, consisting of, or consisting essentially of, a singlechain variable fragment (scFv) of the antigen binding domain of m971.The m971 antibody provides improved binding to CD22 as compared to theHA22 immunotoxin and also binds to a different CD22 epitope. Xiao etal., mAbs 1:3, 297-303 (2009).

The antigen binding domain may comprise a light chain variable regionand/or a heavy chain variable region. In an embodiment of the invention,the heavy chain variable region comprises a CDR1 region, a CDR2 region,and a CDR3 region. In this regard, the antigen binding domain maycomprise one or more of a heavy chain CDR1 region comprising SEQ ID NO:1; a heavy chain CDR2 region comprising SEQ ID NO: 2; and a heavy chainCDR3 region comprising SEQ ID NO: 3. Preferably, the heavy chaincomprises all of SEQ ID NOs: 1-3.

In an embodiment of the invention, the light chain variable region maycomprise a light chain CDR1 region, a light chain CDR2 region, and alight chain CDR3 region. In this regard, the antigen binding domain maycomprise one or more of a light chain CDR1 region comprising SEQ ID NO:4; a light chain CDR2 region comprising SEQ ID NO: 5; and a light chainCDR3 region comprising, SEQ ID NO: 6. Preferably, the light chaincomprises all of SEQ ID NOs: 4-6. In an especially preferred embodiment,the antigen binding domain comprises all of SEQ ID NO: 1-6.

The light chain variable region of the antigen binding domain maycomprise, consist of, or consist essentially of, SEQ ID NO: 7. The heavychain variable region of the antigen binding domain may comprise,consist of, or consist essentially of, SEQ ID NO: 8. Accordingly, in anembodiment of the invention, the antigen binding domain comprises alight chain variable region comprising SEQ ID NO: 7 and/or a heavy chainvariable region comprising SEQ ID NO: 8. Preferably, the antigen bindingdomain comprises both SEQ ID NOs: 7 and 8.

In an embodiment of the invention, the light chain variable region andthe heavy chain variable region may be joined by a linker. The linkermay comprise any suitable amino acid sequence. In an embodiment of theinvention, the linker may comprise, consist, or consist essentially of,SEQ ID NO: 11.

In an embodiment, the antigen binding domain comprises a light chainvariable region and a heavy chain variable region. In this regard, anembodiment of the antigen binding domain comprising both a light chainvariable region and a heavy chain variable region comprises, consistsof, or consists essentially of, SEQ ID NO: 9.

In an embodiment, the antigen binding domain comprises a leadersequence. The leader sequence may be positioned at the amino terminus ofthe light chain variable region. The leader sequence may comprise anysuitable leader sequence. In an embodiment, the leader sequence is ahuman granulocyte-macrophage colony-stimulating factor (GM-CSF) receptorsequence. In this regard, the antigen binding domain comprises a leadersequence comprising, consisting of, or consisting essentially of SEQ IDNO: 10. In an embodiment of the invention, while the leader sequence mayfacilitate expression of the CAR on the surface of the cell, thepresence of the leader sequence in an expressed CAR is not necessary inorder for the CAR to function. In an embodiment of the invention, uponexpression of the CAR on the cell surface, the leader sequence may becleaved off of the CAR. Accordingly, in an embodiment of the invention,the CAR lacks a leader sequence.

In an embodiment of the invention, the CAR comprises a transmembranedomain. In an embodiment of the invention, the transmembrane domaincomprises i) CD8 and/or ii) CD28. In a preferred embodiment, the CD8 andCD28 are human. The CD8 or CD28 may comprise less than the whole CD8 orCD28, respectively. In this regard, the CAR comprises a CD8transmembrane domain comprising, consisting of, or consistingessentially of SEQ ID NO: 12 or 18 and/or a CD28 transmembrane domaincomprising, consisting of, or consisting essentially of SEQ ID NO: 15.

In an embodiment of the invention, the CAR comprises an intracellular Tcell signaling domain comprising any one or more of i) CD28, ii) CD137,and/or iii) CD3 zeta (ζ). In a preferred embodiment, the CD28, CD137,and CD3 zeta are human. CD28 is a T cell marker important in T cellco-stimulation. CD137, also known as 4-1BB, transmits a potentcostimulatory signal to T cells, promoting differentiation and enhancinglong-term survival of T lymphocytes. CD3ζ associates with TCRs toproduce a signal and contains immunoreceptor tyrosine-based activationmotifs (ITAMs). The CD28, CD137, or CD3 zeta may comprise less than thewhole CD28, CD137, or CD3 zeta, respectively. In this regard, theintracellular T cell signaling domain comprises a CD28 amino acidsequence comprising, consisting of, or consisting essentially of, SEQ IDNO: 16 or 19, a CD137 amino acid sequence comprising, consisting of, orconsisting essentially of, SEQ ID NO: 13 or 20, and/or a CD3 zeta aminoacid sequence comprising, consisting of, or consisting essentially of,SEQ ID NO: 14, 17, or 21.

In an embodiment of the invention, the CAR comprises a transmembranedomain comprising CD28 and an intracellular T cell signaling domaincomprising CD28 and CD3 zeta. In this regard, the CAR may comprise eachof SEQ ID NOs: 15-17. Preferably, the CAR comprises a) each of SEQ IDNOs: 1-6 and 15-17; b) 7-8 and 15-17; c) 9 and 15-17; or d) 9-10 and15-17.

In an embodiment of the invention, the CAR comprises a transmembranedomain comprising CD8 and an intracellular T cell signaling domaincomprising CD28, CD137, and CD3 zeta. In this regard, the CAR maycomprise each of SEQ ID NOs: 18-21. Preferably, the CAR comprises a)each of SEQ ID NOs: 1-6 and 18-21; b) 7-8 and 18-21; c) 9 and 18-21; ord) 9-10 and 18-21.

In an embodiment of the invention, the CAR comprises a transmembranedomain comprising CD8 and an intracellular T cell signaling domaincomprising CD137 and CD3 zeta. In this regard, the CAR may comprise eachof SEQ ID NOs: 12-14. Preferably, the CAR comprises a) each of SEQ IDNOs: 1-6 and 12-14; b) 7-8 and 12-14; c) 9 and 12-14; or d) 9-10 and12-14.

Additional embodiments of the invention provide CARs comprising,consisting of, or consisting essentially of any of, the amino acidsequences set forth in Table 1.

TABLE 1 Antigen Binding Transmembrane and SEQ ID NO: Domain SignalingDomains SEQ ID NO: 22 m971 CD8 transmembrane domain (second generation,CD137 and CD3ζ intracellular T cell version 2) signaling domains SEQ IDNO: 23 m971 CD28 transmembrane domain (second generation, CD28 and CD3ζintracellular T cell version 1) signaling domains SEQ ID NO: 24 m971 CD8transmembrane domain (third generation) CD28, CD137, and CD3ζintracellular T cell signaling domains

Included in the scope of the invention are functional portions of theinventive CARs described herein. The term “functional portion” when usedin reference to a CAR refers to any part or fragment of the CAR of theinvention, which part or fragment retains the biological activity of theCAR of which it is a part (the parent CAR). Functional portionsencompass, for example, those parts of a CAR that retain the ability torecognize target cells, or detect, treat, or prevent a disease, to asimilar extent, the same extent, or to a higher extent, as the parentCAR. In reference to the parent CAR, the functional portion cancomprise, for instance, about 10%, 25%, 30%, 50%, 68%, 80%, 90%, 95%, ormore, of the parent CAR.

The functional portion can comprise additional amino acids at the aminoor carboxy terminus of the portion, or at both termini, which additionalamino acids are not found in the amino acid sequence of the parent CAR.Desirably, the additional amino acids do not interfere with thebiological function of the functional portion, e.g., recognize targetcells, detect cancer, treat or prevent cancer, etc. More desirably, theadditional amino acids enhance the biological activity, as compared tothe biological activity of the parent CAR.

Included in the scope of the invention are functional variants of theinventive CARs described herein. The term “functional variant” as usedherein refers to a CAR, polypeptide, or protein having substantial orsignificant sequence identity or similarity to a parent CAR, whichfunctional variant retains the biological activity of the CAR of whichit is a variant. Functional variants encompass, for example, thosevariants of the CAR described herein (the parent CAR) that retain theability to recognize target cells to a similar extent, the same extent,or to a higher extent, as the parent CAR. In reference to the parentCAR, the functional variant can, for instance, be at least about 30%,about 50%, about 75%, about 80%, about 90%, about 98%, about 99% or moreidentical in amino acid sequence to the parent CAR.

A functional variant can, for example, comprise the amino acid sequenceof the parent CAR with at least one conservative amino acidsubstitution. Alternatively or additionally, the functional variants cancomprise the amino acid sequence of the parent CAR with at least onenon-conservative amino acid substitution. In this case, it is preferablefor the non-conservative amino acid substitution to not interfere withor inhibit the biological activity of the functional variant. Thenon-conservative amino acid substitution may enhance the biologicalactivity of the functional variant, such that the biological activity ofthe functional variant is increased as compared to the parent CAR.

Amino acid substitutions of the inventive CARs are preferablyconservative amino acid substitutions. Conservative amino acidsubstitutions are known in the art, and include amino acid substitutionsin which one amino acid having certain physical and/or chemicalproperties is exchanged for another amino acid that has the same orsimilar chemical or physical properties. For instance, the conservativeamino acid substitution can be an acidic/negatively charged polar aminoacid substituted for another acidic/negatively charged polar amino acid(e.g., Asp or Glu), an amino acid with a nonpolar side chain substitutedfor another amino acid with a nonpolar side chain (e.g., Ala, Gly, Val,Ile, Leu, Met, Phe, Pro, Trp, Cys, Val, etc.), a basic/positivelycharged polar amino acid substituted for another basic/positivelycharged polar amino acid (e.g. Lys, His, Arg, etc.), an uncharged aminoacid with a polar side chain substituted for another uncharged aminoacid with a polar side chain (e.g., Asn, Gln, Ser, Thr, Tyr, etc.), anamino acid with a beta-branched side-chain substituted for another aminoacid with a beta-branched side-chain (e.g., Ile, Thr, and Val), an aminoacid with an aromatic side-chain substituted for another amino acid withan aromatic side chain (e.g., His, Phe, Trp, and Tyr), etc.

The CAR can consist essentially of the specified amino acid sequence orsequences described herein, such that other components, e.g., otheramino acids, do not materially change the biological activity of thefunctional variant.

The CARs of embodiments of the invention (including functional portionsand functional variants) can be of any length, i.e., can comprise anynumber of amino acids, provided that the CARs (or functional portions orfunctional variants thereof) retain their biological activity, e.g., theability to specifically bind to antigen, detect diseased cells in amammal, or treat or prevent disease in a mammal, etc. For example, theCAR can be about 50 to about 5000 amino acids long, such as 50, 70, 75,100, 125, 150, 175, 200, 300, 400, 500, 600, 700, 800, 900, 1000 or moreamino acids in length.

The CARs of embodiments of the invention (including functional portionsand functional variants of the invention) can comprise synthetic aminoacids in place of one or more naturally-occurring amino acids. Suchsynthetic amino acids are known in the art, and include, for example,aminocyclohexane carboxylic acid, norleucine, α-amino n-decanoic acid,homoserine, S-acetylaminomethyl-cysteine, trans-3- andtrans-4-hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine,4-chlorophenylalanine, 4-carboxyphenylalanine, β-phenylserine3-hydroxyphenylalanine, phenylglycine, α-naphthylalanine,cyclohexylalanine, cyclohexylglycine, indoline-2-carboxylic acid,1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, aminomalonic acid,aminomalonic acid monoamide, N′-benzyl-N′-methyl-lysine,N′,N′-dibenzyl-lysine, 6-hydroxylysine, omithine, α-aminocyclopentanecarboxylic acid, α-aminocyclohexane carboxylic acid, α-aminocycloheptanecarboxylic acid, α-(2-amino-2-norbornane)-carboxylic acid,α,γ-diaminobutyric acid, α,β-diaminopropionic acid, homophenylalanine,and α-tert-butylglycine.

The CARs of embodiments of the invention (including functional portionsand functional variants) can be glycosylated, amidated, carboxylated,phosphorylated, esterified, N-acylated, cyclized via, e.g., a disulfidebridge, or converted into an acid addition salt and/or optionallydimerized or polymerized, or conjugated.

The CARs of embodiments of the invention (including functional portionsand functional variants thereof) can be obtained by methods known in theart. The CARs may be made by any suitable method of making polypeptidesor proteins. Suitable methods of de novo synthesizing polypeptides andproteins are described in references, such as Chan et al., Fmoc SolidPhase Peptide Synthesis, Oxford University Press, Oxford, UnitedKingdom, 2000; Peptide and Protein Drug Analysis, ed. Reid, R., MarcelDekker, Inc., 2000; Epitope Mapping, ed. Westwood et al., OxfordUniversity Press, Oxford, United Kingdom, 2001; and U.S. Pat. No.5,449,752. Also, polypeptides and proteins can be recombinantly producedusing the nucleic acids described herein using standard recombinantmethods. See, for instance, Sambrook et al., Molecular Cloning: ALaboratory Manual, 3^(rd) ed., Cold Spring Harbor Press, Cold SpringHarbor, N.Y. 2001; and Ausubel et al., Current Protocols in MolecularBiology, Greene Publishing Associates and John Wiley & Sons, NY, 1994.Further, some of the CARs of the invention (including functionalportions and functional variants thereof) can be isolated and/orpurified from a source, such as a plant, a bacterium, an insect, amammal, e.g., a rat, a human, etc. Methods of isolation and purificationare well-known in the art. Alternatively, the CARs described herein(including functional portions and functional variants thereof) can becommercially synthesized by companies, such as Synpep (Dublin, Calif.),Peptide Technologies Corp. (Gaithersburg, Md.), and Multiple PeptideSystems (San Diego, Calif.). In this respect, the inventive CARs can besynthetic, recombinant, isolated, and/or purified.

An embodiment of the invention further provides an antibody, or antigenbinding portion thereof, which specifically binds to an epitope of theCARs of the invention. The antibody can be any type of immunoglobulinthat is known in the art. For instance, the antibody can be of anyisotype, e.g., IgA, IgD, IgE, IgG, IgM, etc. The antibody can bemonoclonal or polyclonal. The antibody can be a naturally-occurringantibody, e.g., an antibody isolated and/or purified from a mammal,e.g., mouse, rabbit, goat, horse, chicken, hamster, human, etc.Alternatively, the antibody can be a genetically-engineered antibody,e.g., a humanized antibody or a chimeric antibody. The antibody can bein monomeric or polymeric form. Also, the antibody can have any level ofaffinity or avidity for the functional portion of the inventive CAR.

Methods of testing antibodies for the ability to bind to any functionalportion of the inventive CAR are known in the art and include anyantibody-antigen binding assay, such as, for example, radioimmunoassay(RIA), ELISA, Western blot, immunoprecipitation, and competitiveinhibition assays (see, e.g., Janeway et al., infra, and U.S. PatentApplication Publication No. 2002/0197266 A1).

Suitable methods of making antibodies are known in the art. Forinstance, standard hybridoma methods are described in, e.g., Kohler andMilstein, Eur. J. Immunol., 5, 511-519 (1976), Harlow and Lane (eds.),Antibodies: A Laboratory Manual, CSH Press (1988), and C. A. Janeway etal. (eds.), Immunobiology, 5^(th) Ed., Garland Publishing, New York,N.Y. (2001)). Alternatively, other methods, such as EBV-hybridomamethods (Haskard and Archer, J. Immunol. Methods, 74(2), 361-67 (1984),and Roder et al., Methods Enzymol., 121, 140-67 (1986)), andbacteriophage vector expression systems (see, e.g., Huse et al.,Science, 246, 1275-81 (1989)) are known in the art. Further, methods ofproducing antibodies in non-human animals are described in, e.g., U.S.Pat. Nos. 5,545,806, 5,569,825, and 5,714,352, and U.S. PatentApplication Publication No. 2002/0197266 A1.

Phage display furthermore can be used to generate an antibody. In thisregard, phage libraries encoding antigen-binding variable (V) domains ofantibodies can be generated using standard molecular biology andrecombinant DNA techniques (see, e.g., Sambrook et al., supra, andAusubel et al., supra). Phage encoding a variable region with thedesired specificity are selected for specific binding to the desiredantigen, and a complete or partial antibody is reconstituted comprisingthe selected variable domain. Nucleic acid sequences encoding thereconstituted antibody are introduced into a suitable cell line, such asa myeloma cell used for hybridoma production, such that antibodieshaving the characteristics of monoclonal antibodies are secreted by thecell (see, e.g., Janeway et al., supra, Huse et al., supra, and U.S.Pat. No. 6,265,150).

Antibodies can be produced by transgenic mice that are transgenic forspecific heavy and light chain immunoglobulin genes. Such methods areknown in the art and described in, for example U.S. Pat. Nos. 5,545,806and 5,569,825, and Janeway et al., supra.

Methods for generating humanized antibodies are well known in the artand are described in detail in, for example, Janeway et al., supra, U.S.Pat. Nos. 5,225,539, 5,585,089 and 5,693,761, European Patent No.0239400 B1, and United Kingdom Patent No. 2188638. Humanized antibodiescan also be generated using the antibody resurfacing technologydescribed in U.S. Pat. No. 5,639,641 and Pedersen et al., J. Mol. Biol.,235, 959-973 (1994).

An embodiment of the invention also provides antigen binding portions ofany of the antibodies described herein. The antigen binding portion canbe any portion that has at least one antigen binding site, such as Fab,F(ab′)₂, dsFv, sFv, diabodies, and triabodies.

A single-chain variable region fragment (sFv) antibody fragment, whichis a truncated Fab fragment including the variable (V) domain of anantibody heavy chain linked to a V domain of a light antibody chain viaa synthetic peptide, can be generated using routine recombinant DNAtechnology techniques (see, e.g., Janeway et al., supra). Similarly,disulfide-stabilized variable region fragments (dsFv) can be prepared byrecombinant DNA technology (see, e.g., Reiter et al., ProteinEngineering, 7, 697-704 (1994)). Antibody fragments of the invention,however, are not limited to these exemplary types of antibody fragments.

Also, the antibody, or antigen binding portion thereof, can be modifiedto comprise a detectable label, such as, for instance, a radioisotope, afluorophore (e.g., fluorescein isothiocyanate (FITC), phycoerythrin(PE)), an enzyme (e.g., alkaline phosphatase, horseradish peroxidase),and element particles (e.g., gold particles).

Further provided by an embodiment of the invention is a nucleic acidcomprising a nucleotide sequence encoding any of the CARs describedherein (including functional portions and functional variants thereof).The nucleic acids of the invention may comprise a nucleotide sequenceencoding any of the leader sequences, antigen binding domains,transmembrane domains, and/or intracellular T cell signaling domainsdescribed herein.

An embodiment of the invention provides a nucleic acid comprising anucleotide sequence encoding a leader sequence and an antigen bindingdomain (including a light chain variable region and a heavy chainvariable region. In this regard, the nucleic acid may comprise, consistof, or consist essentially of, SEQ ID NO: 25.

The nucleic acids of the invention may comprise a nucleotide sequenceencoding any of the transmembrane domains and/or intracellular T cellsignaling domains described herein. An embodiment of the inventionprovides a nucleic acid comprising a nucleotide sequence encoding atransmembrane domain comprising CD28, an intracellular T cell signalingdomain comprising CD28, and an intracellular T cell signaling domaincomprising CD3ζ. In this regard, the nucleic acid may comprise, consistof, or consist essentially of, SEQ ID NO: 27. Another embodiment of theinvention provides a nucleic acid comprising a nucleotide sequenceencoding a transmembrane domain comprising CD8, an intracellular T cellsignaling domain comprising CD28, an intracellular T cell signalingdomain comprising CD137, and an intracellular T cell signaling domaincomprising CD3ζ. In this regard, the nucleic acid may comprise, consistof, or consist essentially of, SEQ ID NO: 28. Still another embodimentof the invention provides a nucleic acid comprising a nucleotidesequence encoding a transmembrane domain comprising CD8, anintracellular T cell signaling domain comprising CD137, and anintracellular T cell signaling domain comprising CD3ζ. In this regard,the nucleic acid may comprise, consist of, or consist essentially of,SEQ ID NO: 26.

In an embodiment of the invention, the nucleic acid comprises anucleotide sequence that encodes a leader sequence, an antigen bindingdomain (including a light chain variable region and a heavy chainvariable region), a transmembrane domain comprising CD28, anintracellular T cell signaling domain comprising CD28, and anintracellular T cell signaling domain comprising CD3ζ. In this regard,the nucleic acid may comprise, consist of, or consist essentially of,both SEQ ID NOs: 25 and 27.

In an embodiment of the invention, the nucleic acid comprises anucleotide sequence that encodes a leader sequence, an antigen bindingdomain (including a light chain variable region and a heavy chainvariable region), a transmembrane domain comprising CD8, anintracellular T cell signaling domain comprising CD28, an intracellularT cell signaling domain comprising CD137, and an intracellular T cellsignaling domain comprising CD3ζ. In this regard, the nucleic acid maycomprise, consist of, or consist essentially of, both SEQ ID NOs: 25 and28.

In an embodiment, the nucleic acid comprises a nucleotide sequence thatencodes a leader sequence, an antigen binding domain (including a lightchain variable region and a heavy chain variable region), atransmembrane domain comprising CD8, an intracellular T cell signalingdomain comprising CD137, and an intracellular T cell signaling domaincomprising CD3ζ. In this regard, the nucleic acid may comprise, consistof, or consist essentially of, both SEQ ID NOs: 25 and 26.

“Nucleic acid” as used herein includes “polynucleotide,”“oligonucleotide,” and “nucleic acid molecule,” and generally means apolymer of DNA or RNA, which can be single-stranded or double-stranded,synthesized or obtained (e.g., isolated and/or purified) from naturalsources, which can contain natural, non-natural or altered nucleotides,and which can contain a natural, non-natural or altered internucleotidelinkage, such as a phosphoroamidate linkage or a phosphorothioatelinkage, instead of the phosphodiester found between the nucleotides ofan unmodified oligonucleotide. In some embodiments, the nucleic aciddoes not comprise any insertions, deletions, inversions, and/orsubstitutions. However, it may be suitable in some instances, asdiscussed herein, for the nucleic acid to comprise one or moreinsertions, deletions, inversions, and/or substitutions. In someembodiments, the nucleic acid may encode additional amino acid sequencesthat do not affect the function of the CAR and which may or may not betranslated upon expression of the nucleic acid by a host cell.

The nucleic acids of an embodiment of the invention may be recombinant.As used herein, the term “recombinant” refers to (i) molecules that areconstructed outside living cells by joining natural or synthetic nucleicacid segments to nucleic acid molecules that can replicate in a livingcell, or (ii) molecules that result from the replication of thosedescribed in (i) above. For purposes herein, the replication can be invitro replication or in vivo replication.

A recombinant nucleic acid may be one that has a sequence that is notnaturally occurring or has a sequence that is made by an artificialcombination of two otherwise separated segments of sequence. Thisartificial combination is often accomplished by chemical synthesis or,more commonly, by the artificial manipulation of isolated segments ofnucleic acids, e.g., by genetic engineering techniques, such as thosedescribed in Sambrook et al., supra. The nucleic acids can beconstructed based on chemical synthesis and/or enzymatic ligationreactions using procedures known in the art. See, for example, Sambrooket al., supra, and Ausubel et al., supra. For example, a nucleic acidcan be chemically synthesized using naturally occurring nucleotides orvariously modified nucleotides designed to increase the biologicalstability of the molecules or to increase the physical stability of theduplex formed upon hybridization (e.g., phosphorothioate derivatives andacridine substituted nucleotides). Examples of modified nucleotides thatcan be used to generate the nucleic acids include, but are not limitedto, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxymethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N⁶-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N⁶-substitutedadenine, 7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N⁶-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, 3-(3-amino-3-N-2-carboxypropyl)uracil, and 2,6-diaminopurine. Alternatively, one or more of the nucleicacids of the invention can be purchased from companies, such asMacromolecular Resources (Fort Collins, Colo.) and Synthegen (Houston,Tex.).

The nucleic acid can comprise any isolated or purified nucleotidesequence which encodes any of the CARs or functional portions orfunctional variants thereof. Alternatively, the nucleotide sequence cancomprise a nucleotide sequence which is degenerate to any of thesequences or a combination of degenerate sequences.

An embodiment of the invention also provides an isolated or purifiednucleic acid comprising a nucleotide sequence which is complementary tothe nucleotide sequence of any of the nucleic acids described herein ora nucleotide sequence which hybridizes under stringent conditions to thenucleotide sequence of any of the nucleic acids described herein.

The nucleotide sequence which hybridizes under stringent conditions mayhybridize under high stringency conditions. By “high stringencyconditions” is meant that the nucleotide sequence specificallyhybridizes to a target sequence (the nucleotide sequence of any of thenucleic acids described herein) in an amount that is detectably strongerthan non-specific hybridization. High stringency conditions includeconditions which would distinguish a polynucleotide with an exactcomplementary sequence, or one containing only a few scatteredmismatches from a random sequence that happened to have a few smallregions (e.g., 3-10 bases) that matched the nucleotide sequence. Suchsmall regions of complementarity are more easily melted than afull-length complement of 14-17 or more bases, and high stringencyhybridization makes them easily distinguishable. Relatively highstringency conditions would include, for example, low salt and/or hightemperature conditions, such as provided by about 0.02-0.1 M NaCl or theequivalent, at temperatures of about 50-70° C. Such high stringencyconditions tolerate little, if any, mismatch between the nucleotidesequence and the template or target strand, and are particularlysuitable for detecting expression of any of the inventive CARs. It isgenerally appreciated that conditions can be rendered more stringent bythe addition of increasing amounts of formamide.

The invention also provides a nucleic acid comprising a nucleotidesequence that is at least about 70% or more, e.g., about 80%, about 90%,about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about97%, about 98%, or about 99% identical to any of the nucleic acidsdescribed herein.

In an embodiment, the nucleic acids of the invention can be incorporatedinto a recombinant expression vector. In this regard, an embodiment ofthe invention provides recombinant expression vectors comprising any ofthe nucleic acids of the invention. For purposes herein, the term“recombinant expression vector” means a genetically-modifiedoligonucleotide or polynucleotide construct that permits the expressionof an mRNA, protein, polypeptide, or peptide by a host cell, when theconstruct comprises a nucleotide sequence encoding the mRNA, protein,polypeptide, or peptide, and the vector is contacted with the cell underconditions sufficient to have the mRNA, protein, polypeptide, or peptideexpressed within the cell. The vectors of the invention are notnaturally-occurring as a whole. However, parts of the vectors can benaturally-occurring. The inventive recombinant expression vectors cancomprise any type of nucleotides, including, but not limited to DNA andRNA, which can be single-stranded or double-stranded, synthesized orobtained in part from natural sources, and which can contain natural,non-natural or altered nucleotides. The recombinant expression vectorscan comprise naturally-occurring or non-naturally-occurringinternucleotide linkages, or both types of linkages. Preferably, thenon-naturally occurring or altered nucleotides or internucleotidelinkages do not hinder the transcription or replication of the vector.

In an embodiment, the recombinant expression vector of the invention canbe any suitable recombinant expression vector, and can be used totransform or transfect any suitable host cell. Suitable vectors includethose designed for propagation and expansion or for expression or both,such as plasmids and viruses. The vector can be selected from the groupconsisting of the pUC series (Fermentas Life Sciences, Glen Bumie, Md.),the pBluescript series (Stratagene, LaJolla, Calif.), the pET series(Novagen, Madison, Wis.), the pGEX series (Pharmacia Biotech, Uppsala,Sweden), and the pEX series (Clontech, Palo Alto, Calif.). Bacteriophagevectors, such as XGT10, XGT11, XZapII (Stratagene), XEMBL4, and XNM1149,also can be used. Examples of plant expression vectors include pBIO1,pBI101.2, pBI101.3, pBI121 and pBIN19 (Clontech). Examples of animalexpression vectors include pEUK-C1, pMAM, and pMAMneo (Clontech). Therecombinant expression vector may be a viral vector, e.g., a retroviralvector or a lentiviral vector. In some embodiments, the vector can be atransposon.

A number of transfection techniques are generally known in the art (see,e.g., Graham et al., Virology, 52: 456-467 (1973); Sambrook et al.,supra; Davis et al., Basic Methods in Molecular Biology, Elsevier(1986); and Chu et al., Gene, 13: 97 (1981). Transfection methodsinclude calcium phosphate co-precipitation (see, e.g., Graham et al.,supra), direct micro injection into cultured cells (see, e.g., Capecchi,Cell, 22: 479-488 (1980)), electroporation (see, e.g., Shigekawa et al.,BioTechniques, 6: 742-751 (1988)), liposome mediated gene transfer (see,e.g., Mannino et al., BioTechniques, 6: 682-690 (1988)), lipid mediatedtransduction (see, e.g., Felgner et al., Proc. Natl. Acad. Sci. USA, 84:7413-7417 (1987)), and nucleic acid delivery using high velocitymicroprojectiles (see, e.g., Klein et al., Nature, 327: 70-73 (1987)).

In an embodiment, the recombinant expression vectors of the inventioncan be prepared using standard recombinant DNA techniques described in,for example, Sambrook et al., supra, and Ausubel et al., supra.Constructs of expression vectors, which are circular or linear, can beprepared to contain a replication system functional in a prokaryotic oreukaryotic host cell. Replication systems can be derived, e.g., fromColE1, 2μ plasmid, λ, SV40, bovine papilloma virus, and the like.

The recombinant expression vector may comprise regulatory sequences,such as transcription and translation initiation and termination codons,which are specific to the type of host cell (e.g., bacterium, fungus,plant, or animal) into which the vector is to be introduced, asappropriate, and taking into consideration whether the vector is DNA- orRNA-based. Examples of sequences including termination codons includeSEQ ID NOs: 32-33. The recombinant expression vector may compriserestriction sites to facilitate cloning. Examples of sequences includingrestriction sites include SEQ ID NOs: 29-31.

The recombinant expression vector can include one or more marker genes,which allow for selection of transformed or transfected host cells.Marker genes include biocide resistance, e.g., resistance toantibiotics, heavy metals, etc., complementation in an auxotrophic hostto provide prototrophy, and the like. Suitable marker genes for theinventive expression vectors include, for instance, neomycin/G418resistance genes, hygromycin resistance genes, histidinol resistancegenes, tetracycline resistance genes, and ampicillin resistance genes.

The recombinant expression vector can comprise a native or nonnativepromoter operably linked to the nucleotide sequence encoding the CAR(including functional portions and functional variants thereof), or tothe nucleotide sequence which is complementary to or which hybridizes tothe nucleotide sequence encoding the CAR. The selection of promoters,e.g., strong, weak, inducible, tissue-specific anddevelopmental-specific, is within the ordinary skill of the artisan.Similarly, the combining of a nucleotide sequence with a promoter isalso within the skill of the artisan. The promoter can be a non-viralpromoter or a viral promoter, e.g., a cytomegalovirus (CMV) promoter, anSV40 promoter, an RSV promoter, or a promoter found in the long-terminalrepeat of the murine stem cell virus.

The inventive recombinant expression vectors can be designed for eithertransient expression, for stable expression, or for both. Also, therecombinant expression vectors can be made for constitutive expressionor for inducible expression.

Further, the recombinant expression vectors can be made to include asuicide gene. As used herein, the term “suicide gene” refers to a genethat causes the cell expressing the suicide gene to die. The suicidegene can be a gene that confers sensitivity to an agent, e.g., a drug,upon the cell in which the gene is expressed, and causes the cell to diewhen the cell is contacted with or exposed to the agent. Suicide genesare known in the art (see, for example, Suicide Gene Therapy: Methodsand Reviews, Springer, Caroline J. (Cancer Research UK Centre for CancerTherapeutics at the Institute of Cancer Research, Sutton, Surrey, UK),Humana Press, 2004) and include, for example, the Herpes Simplex Virus(HSV) thymidine kinase (TK) gene, cytosine daminase, purine nucleosidephosphorylase, and nitroreductase.

Included in the scope of the invention are conjugates, e.g.,bioconjugates, comprising any of the inventive CARs (including any ofthe functional portions or variants thereof), nucleic acids, recombinantexpression vectors, host cells, populations of host cells, orantibodies, or antigen binding portions thereof. Conjugates, as well asmethods of synthesizing conjugates in general, are known in the art(See, for instance, Hudecz, F., Methods Mol. Biol. 298: 209-223 (2005)and Kirin et al., Inorg Chem. 44(15): 5405-5415 (2005)).

An embodiment of the invention further provides a host cell comprisingany of the recombinant expression vectors described herein. As usedherein, the term “host cell” refers to any type of cell that can containthe inventive recombinant expression vector. The host cell can be aeukaryotic cell, e.g., plant, animal, fungi, or algae, or can be aprokaryotic cell, e.g., bacteria or protozoa. The host cell can be acultured cell or a primary cell, i.e., isolated directly from anorganism, e.g., a human. The host cell can be an adherent cell or asuspended cell, i.e., a cell that grows in suspension. Suitable hostcells are known in the art and include, for instance, DH5a E. colicells, Chinese hamster ovarian cells, monkey VERO cells, COS cells,HEK293 cells, and the like. For purposes of amplifying or replicatingthe recombinant expression vector, the host cell may be a prokaryoticcell, e.g., a DH5a cell. For purposes of producing a recombinant CAR,the host cell may be a mammalian cell. The host cell may be a humancell. While the host cell can be of any cell type, can originate fromany type of tissue, and can be of any developmental stage, the host cellmay be a peripheral blood lymphocyte (PBL) or a peripheral bloodmononuclear cell (PBMC). The host cell may be a T cell.

For purposes herein, the T cell can be any T cell, such as a cultured Tcell, e.g., a primary T cell, or a T cell from a cultured T cell line,e.g., Jurkat, SupT1, etc., or a T cell obtained from a mammal. Ifobtained from a mammal, the T cell can be obtained from numeroussources, including but not limited to blood, bone marrow, lymph node,the thymus, or other tissues or fluids. T cells can also be enriched foror purified. The T cell may be a human T cell. The T cell may be a Tcell isolated from a human. The T cell can be any type of T cell and canbe of any developmental stage, including but not limited to, CD4⁺/CD8⁺double positive T cells, CD4⁺ helper T cells, e.g., Th₁ and Th₂ cells,CD8⁺ T cells (e.g., cytotoxic T cells), tumor infiltrating cells, memoryT cells, naïve T cells, and the like. The T cell may be a CD8⁺ T cell ora CD4⁺ T cell.

Also provided by an embodiment of the invention is a population of cellscomprising at least one host cell described herein. The population ofcells can be a heterogeneous population comprising the host cellcomprising any of the recombinant expression vectors described, inaddition to at least one other cell, e.g., a host cell (e.g., a T cell),which does not comprise any of the recombinant expression vectors, or acell other than a T cell, e.g., a B cell, a macrophage, a neutrophil, anerythrocyte, a hepatocyte, an endothelial cell, an epithelial cell, amuscle cell, a brain cell, etc. Alternatively, the population of cellscan be a substantially homogeneous population, in which the populationcomprises mainly host cells (e.g., consisting essentially of) comprisingthe recombinant expression vector. The population also can be a clonalpopulation of cells, in which all cells of the population are clones ofa single host cell comprising a recombinant expression vector, such thatall cells of the population comprise the recombinant expression vector.In one embodiment of the invention, the population of cells is a clonalpopulation comprising host cells comprising a recombinant expressionvector as described herein.

CARs (including functional portions and variants thereof), nucleicacids, recombinant expression vectors, host cells (including populationsthereof), and antibodies (including antigen binding portions thereof),all of which are collectively referred to as “inventive CAR materials”hereinafter, can be isolated and/or purified. The term “isolated” asused herein means having been removed from its natural environment. Theterm “purified” or “isolated” does not require absolute purity orisolation; rather, it is intended as a relative term. Thus, for example,a purified (or isolated) host cell preparation is one in which the hostcell is more pure than cells in their natural environment within thebody. Such host cells may be produced, for example, by standardpurification techniques. In some embodiments, a preparation of a hostcell is purified such that the host cell represents at least about 50%,for example at least about 70%, of the total cell content of thepreparation. For example, the purity can be at least about 50%, can begreater than about 60%, about 70% or about 80%, or can be about 100%.

The inventive CAR materials can be formulated into a composition, suchas a pharmaceutical composition. In this regard, an embodiment of theinvention provides a pharmaceutical composition comprising any of theCARs, functional portions, functional variants, nucleic acids,expression vectors, host cells (including populations thereof), andantibodies (including antigen binding portions thereof), and apharmaceutically acceptable carrier. The inventive pharmaceuticalcompositions containing any of the inventive CAR materials can comprisemore than one inventive CAR material, e.g., a CAR and a nucleic acid, ortwo or more different CARs. Alternatively, the pharmaceuticalcomposition can comprise an inventive CAR material in combination withother pharmaceutically active agents or drugs, such as chemotherapeuticagents, e.g., asparaginase, busulfan, carboplatin, cisplatin,daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea,methotrexate, paclitaxel, rituximab, vinblastine, vincristine, etc. In apreferred embodiment, the pharmaceutical composition comprises theinventive host cell or populations thereof.

The inventive CAR materials can be provided in the form of a salt, e.g.,a pharmaceutically acceptable salt. Suitable pharmaceutically acceptableacid addition salts include those derived from mineral acids, such ashydrochloric, hydrobromic, phosphoric, metaphosphoric, nitric, andsulphuric acids, and organic acids, such as tartaric, acetic, citric,malic, lactic, fumaric, benzoic, glycolic, gluconic, succinic, andarylsulphonic acids, for example, p-toluenesulphonic acid.

With respect to pharmaceutical compositions, the pharmaceuticallyacceptable carrier can be any of those conventionally used and islimited only by chemico-physical considerations, such as solubility andlack of reactivity with the active agent(s), and by the route ofadministration. The pharmaceutically acceptable carriers describedherein, for example, vehicles, adjuvants, excipients, and diluents, arewell-known to those skilled in the art and are readily available to thepublic. It is preferred that the pharmaceutically acceptable carrier beone which is chemically inert to the active agent(s) and one which hasno detrimental side effects or toxicity under the conditions of use.

The choice of carrier will be determined in part by the particularinventive CAR material, as well as by the particular method used toadminister the inventive CAR material. Accordingly, there are a varietyof suitable formulations of the pharmaceutical composition of theinvention. Preservatives may be used. Suitable preservatives mayinclude, for example, methylparaben, propylparaben, sodium benzoate, andbenzalkonium chloride. A mixture of two or more preservatives optionallymay be used. The preservative or mixtures thereof are typically presentin an amount of about 0.0001% to about 2% by weight of the totalcomposition.

Suitable buffering agents may include, for example, citric acid, sodiumcitrate, phosphoric acid, potassium phosphate, and various other acidsand salts. A mixture of two or more buffering agents optionally may beused. The buffering agent or mixtures thereof are typically present inan amount of about 0.001% to about 4% by weight of the totalcomposition.

The concentration of inventive CAR material in the pharmaceuticalformulations can vary, e.g., from less than about 1%, usually at or atleast about 10%, to as much as about 20% to about 50% or more by weight,and can be selected primarily by fluid volumes, and viscosities, inaccordance with the particular mode of administration selected.

Methods for preparing administrable (e.g., parenterally administrable)compositions are known or apparent to those skilled in the art and aredescribed in more detail in, for example, Remington: The Science andPractice of Pharmacy, Lippincott Williams & Wilkins; 21st ed. (May 1,2005).

The following formulations for oral, aerosol, parenteral (e.g.,subcutaneous, intravenous, intraarterial, intramuscular, intradermal,interperitoneal, and intrathecal), and topical administration are merelyexemplary and are in no way limiting. More than one route can be used toadminister the inventive CAR materials, and in certain instances, aparticular route can provide a more immediate and more effectiveresponse than another route.

Formulations suitable for oral administration can comprise or consist of(a) liquid solutions, such as an effective amount of the inventive CARmaterial dissolved in diluents, such as water, saline, or orange juice;(b) capsules, sachets, tablets, lozenges, and troches, each containing apredetermined amount of the active ingredient, as solids or granules;(c) powders; (d) suspensions in an appropriate liquid; and (e) suitableemulsions. Liquid formulations may include diluents, such as water andalcohols, for example, ethanol, benzyl alcohol, and the polyethylenealcohols, either with or without the addition of a pharmaceuticallyacceptable surfactant. Capsule forms can be of the ordinary hard orsoftshelled gelatin type containing, for example, surfactants,lubricants, and inert fillers, such as lactose, sucrose, calciumphosphate, and corn starch. Tablet forms can include one or more oflactose, sucrose, mannitol, corn starch, potato starch, alginic acid,microcrystalline cellulose, acacia, gelatin, guar gum, colloidal silicondioxide, croscarmellose sodium, talc, magnesium stearate, calciumstearate, zinc stearate, stearic acid, and other excipients, colorants,diluents, buffering agents, disintegrating agents, moistening agents,preservatives, flavoring agents, and other pharmacologically compatibleexcipients. Lozenge forms can comprise the inventive CAR material in aflavor, usually sucrose and acacia or tragacanth, as well as pastillescomprising the inventive CAR material in an inert base, such as gelatinand glycerin, or sucrose and acacia, emulsions, gels, and the likecontaining, in addition to, such excipients as are known in the art.

Formulations suitable for parenteral administration include aqueous andnonaqueous isotonic sterile injection solutions, which can containantioxidants, buffers, bacteriostats, and solutes that render theformulation isotonic with the blood of the intended recipient, andaqueous and nonaqueous sterile suspensions that can include suspendingagents, solubilizers, thickening agents, stabilizers, and preservatives.The inventive CAR material can be administered in a physiologicallyacceptable diluent in a pharmaceutical carrier, such as a sterile liquidor mixture of liquids, including water, saline, aqueous dextrose andrelated sugar solutions, an alcohol, such as ethanol or hexadecylalcohol, a glycol, such as propylene glycol or polyethylene glycol,dimethylsulfoxide, glycerol, ketals such as2,2-dimethyl-1,3-dioxolane-4-methanol, ethers, poly(ethyleneglycol) 400,oils, fatty acids, fatty acid esters or glycerides, or acetylated fattyacid glycerides with or without the addition of a pharmaceuticallyacceptable surfactant, such as a soap or a detergent, suspending agent,such as pectin, carbomers, methylcellulose,hydroxypropylmethylcellulose, or carboxymethylcellulose, or emulsifyingagents and other pharmaceutical adjuvants.

Oils, which can be used in parenteral formulations include petroleum,animal, vegetable, or synthetic oils. Specific examples of oils includepeanut, soybean, sesame, cottonseed, corn, olive, petrolatum, andmineral. Suitable fatty acids for use in parenteral formulations includeoleic acid, stearic acid, and isostearic acid. Ethyl oleate andisopropyl myristate are examples of suitable fatty acid esters.

Suitable soaps for use in parenteral formulations include fatty alkalimetal, ammonium, and triethanolamine salts, and suitable detergentsinclude (a) cationic detergents such as, for example, dimethyl dialkylammonium halides, and alkyl pyridinium halides, (b) anionic detergentssuch as, for example, alkyl, aryl, and olefin sulfonates, alkyl, olefin,ether, and monoglyceride sulfates, and sulfosuccinates, (c) nonionicdetergents such as, for example, fatty amine oxides, fatty acidalkanolamides, and polyoxyethylenepolypropylene copolymers, (d)amphoteric detergents such as, for example, alkyl-β-aminopropionates,and 2-alkyl-imidazoline quaternary ammonium salts, and (e) mixturesthereof.

The parenteral formulations will typically contain, for example, fromabout 0.5% to about 25% by weight of the inventive CAR material insolution. Preservatives and buffers may be used. In order to minimize oreliminate irritation at the site of injection, such compositions maycontain one or more nonionic surfactants having, for example, ahydrophile-lipophile balance (HLB) of from about 12 to about 17. Thequantity of surfactant in such formulations will typically range, forexample, from about 5% to about 15% by weight. Suitable surfactantsinclude polyethylene glycol sorbitan fatty acid esters, such as sorbitanmonooleate and the high molecular weight adducts of ethylene oxide witha hydrophobic base, formed by the condensation of propylene oxide withpropylene glycol. The parenteral formulations can be presented inunit-dose or multi-dose sealed containers, such as ampoules and vials,and can be stored in a freeze-dried (lyophilized) condition requiringonly the addition of the sterile liquid excipient, for example, water,for injections, immediately prior to use. Extemporaneous injectionsolutions and suspensions can be prepared from sterile powders,granules, and tablets of the kind previously described.

Injectable formulations are in accordance with an embodiment of theinvention. The requirements for effective pharmaceutical carriers forinjectable compositions are well-known to those of ordinary skill in theart (see, e.g., Pharmaceutics and Pharmacy Practice, J. B. LippincottCompany, Philadelphia, Pa., Banker and Chalmers, eds., pages 238-250(1982), and ASHP Handbook on Injectable Drugs, Toissel, 4th ed., pages622-630 (1986)).

Topical formulations, including those that are useful for transdermaldrug release, are well known to those of skill in the art and aresuitable in the context of embodiments of the invention for applicationto skin. The inventive CAR material, alone or in combination with othersuitable components, can be made into aerosol formulations to beadministered via inhalation. These aerosol formulations can be placedinto pressurized acceptable propellants, such asdichlorodifluoromethane, propane, nitrogen, and the like. They also maybe formulated as pharmaceuticals for non-pressured preparations, such asin a nebulizer or an atomizer. Such spray formulations also may be usedto spray mucosa.

An “effective amount” or “an amount effective to treat” refers to a dosethat is adequate to prevent or treat cancer in an individual. Amountseffective for a therapeutic or prophylactic use will depend on, forexample, the stage and severity of the disease or disorder beingtreated, the age, weight, and general state of health of the patient,and the judgment of the prescribing physician. The size of the dose willalso be determined by the active selected, method of administration,timing and frequency of administration, the existence, nature, andextent of any adverse side-effects that might accompany theadministration of a particular active, and the desired physiologicaleffect. It will be appreciated by one of skill in the art that variousdiseases or disorders could require prolonged treatment involvingmultiple administrations, perhaps using the inventive CAR materials ineach or various rounds of administration. By way of example and notintending to limit the invention, the dose of the inventive CAR materialcan be about 0.001 to about 1000 mg/kg body weight of the subject beingtreated/day, from about 0.01 to about 10 mg/kg body weight/day, about0.01 mg to about 1 mg/kg body weight/day. When the inventive CARmaterial is a host cell, an exemplary dose of host cells may be aminimum of one million cells (1 mg cells/dose). When the inventive CARmaterial is a nucleic acid packaged in a virus, an exemplary dose ofvirus may be 1 ng/dose.

For purposes of the invention, the amount or dose of the inventive CARmaterial administered should be sufficient to effect a therapeutic orprophylactic response in the subject or animal over a reasonable timeframe. For example, the dose of the inventive CAR material should besufficient to bind to antigen, or detect, treat or prevent disease in aperiod of from about 2 hours or longer, e.g., about 12 to about 24 ormore hours, from the time of administration. In certain embodiments, thetime period could be even longer. The dose will be determined by theefficacy of the particular inventive CAR material and the condition ofthe animal (e.g., human), as well as the body weight of the animal(e.g., human) to be treated.

For purposes of the invention, an assay, which comprises, for example,comparing the extent to which target cells are lysed and/or IFN-γ issecreted by T cells expressing the inventive CAR upon administration ofa given dose of such T cells to a mammal, among a set of mammals ofwhich is each given a different dose of the T cells, could be used todetermine a starting dose to be administered to a mammal. The extent towhich target cells are lysed and/or IFN-γ is secreted uponadministration of a certain dose can be assayed by methods known in theart.

In addition to the aforedescribed pharmaceutical compositions, theinventive CAR materials can be formulated as inclusion complexes, suchas cyclodextrin inclusion complexes, or liposomes. Liposomes can serveto target the inventive CAR materials to a particular tissue. Liposomesalso can be used to increase the half-life of the inventive CARmaterials. Many methods are available for preparing liposomes, asdescribed in, for example, Szoka et al., Ann. Rev. Biophys. Bioeng., 9,467 (1980) and U.S. Pat. Nos. 4,235,871, 4,501,728, 4,837,028, and5,019,369.

The delivery systems useful in the context of embodiments of theinvention may include time-released, delayed release, and sustainedrelease delivery systems such that the delivery of the inventivecomposition occurs prior to, and with sufficient time to cause,sensitization of the site to be treated. The inventive composition canbe used in conjunction with other therapeutic agents or therapies. Suchsystems can avoid repeated administrations of the inventive composition,thereby increasing convenience to the subject and the physician, and maybe particularly suitable for certain composition embodiments of theinvention.

Many types of release delivery systems are available and known to thoseof ordinary skill in the art. They include polymer base systems such aspoly(lactide-glycolide), copolyoxalates, polycaprolactones,polyesteramides, polyorthoesters, polyhydroxybutyric acid, andpolyanhydrides. Microcapsules of the foregoing polymers containing drugsare described in, for example, U.S. Pat. No. 5,075,109. Delivery systemsalso include non-polymer systems that are lipids including sterols suchas cholesterol, cholesterol esters, and fatty acids or neutral fats suchas mono- di- and tri-glycerides; hydrogel release systems; sylasticsystems; peptide based systems; wax coatings; compressed tablets usingconventional binders and excipients; partially fused implants; and thelike. Specific examples include, but are not limited to: (a) erosionalsystems in which the active composition is contained in a form within amatrix such as those described in U.S. Pat. Nos. 4,452,775, 4,667,014,4,748,034, and 5,239,660 and (b) diffusional systems in which an activecomponent permeates at a controlled rate from a polymer such asdescribed in U.S. Pat. Nos. 3,832,253 and 3,854,480. In addition,pump-based hardware delivery systems can be used, some of which areadapted for implantation.

One of ordinary skill in the art will readily appreciate that theinventive CAR materials of the invention can be modified in any numberof ways, such that the therapeutic or prophylactic efficacy of theinventive CAR materials is increased through the modification. Forinstance, the inventive CAR materials can be conjugated either directlyor indirectly through a linker to a targeting moiety. The practice ofconjugating compounds, e.g., inventive CAR materials, to targetingmoieties is known in the art. See, for instance, Wadwa et al., J. DrugTargeting 3: 111 (1995) and U.S. Pat. No. 5,087,616.

Alternatively, the inventive CAR materials can be modified into a depotform, such that the manner in which the inventive CAR materials isreleased into the body to which it is administered is controlled withrespect to time and location within the body (see, for example, U.S.Pat. No. 4,450,150). Depot forms of inventive CAR materials can be, forexample, an implantable composition comprising the inventive CARmaterials and a porous or non-porous material, such as a polymer,wherein the inventive CAR materials are encapsulated by or diffusedthroughout the material and/or degradation of the non-porous material.The depot is then implanted into the desired location within the bodyand the inventive CAR materials are released from the implant at apredetermined rate.

When the inventive CAR materials are administered with one or moreadditional therapeutic agents, one or more additional therapeutic agentscan be coadministered to the mammal. By “coadministering” is meantadministering one or more additional therapeutic agents and theinventive CAR materials sufficiently close in time such that theinventive CAR materials can enhance the effect of one or more additionaltherapeutic agents, or vice versa. In this regard, the inventive CARmaterials can be administered first and the one or more additionaltherapeutic agents can be administered second, or vice versa.Alternatively, the inventive CAR materials and the one or moreadditional therapeutic agents can be administered simultaneously. Anexemplary therapeutic agent that can be co-administered with the CARmaterials is IL-2. It is believed that IL-2 enhances the therapeuticeffect of the inventive CAR materials. For purposes of the inventivemethods, wherein host cells or populations of cells are administered tothe mammal, the cells can be cells that are allogeneic or autologous tothe mammal.

It is contemplated that the inventive pharmaceutical compositions, CARs,nucleic acids, recombinant expression vectors, host cells, orpopulations of cells can be used in methods of treating or preventing adisease in a mammal. Without being bound to a particular theory ormechanism, the inventive CARs have biological activity, e.g., ability torecognize antigen, e.g., CD22, such that the CAR when expressed by acell is able to mediate an immune response against the cell expressingthe antigen, e.g., CD22, for which the CAR is specific. In this regard,an embodiment of the invention provides a method of treating orpreventing cancer in a mammal, comprising administering to the mammalthe CARs, the nucleic acids, the recombinant expression vectors, thehost cells, the population of cells, the antibodies and/or the antigenbinding portions thereof, and/or the pharmaceutical compositions of theinvention in an amount effective to treat or prevent cancer in themammal.

An embodiment of the invention further comprises lymphodepleting themammal prior to administering the inventive CAR materials. Examples oflymphodepletion include, but may not be limited to, nonmyeloablativelymphodepleting chemotherapy, myeloablative lymphodepletingchemotherapy, total body irradiation, etc.

For purposes of the inventive methods, wherein host cells or populationsof cells are administered, the cells can be cells that are allogeneic orautologous to the mammal. Preferably, the cells are autologous to themammal.

The mammal referred to herein can be any mammal. As used herein, theterm “mammal” refers to any mammal, including, but not limited to,mammals of the order Rodentia, such as mice and hamsters, and mammals ofthe order Logomorpha, such as rabbits. The mammals may be from the orderCamivora, including Felines (cats) and Canines (dogs). The mammals maybe from the order Artiodactyla, including Bovines (cows) and Swines(pigs) or of the order Perssodactyla, including Equines (horses). Themammals may be of the order Primates, Ceboids, or Simoids (monkeys) orof the order Anthropoids (humans and apes). Preferably, the mammal is ahuman.

With respect to the inventive methods, the cancer can be any cancer,including any of acute lymphocytic cancer, acute myeloid leukemia,alveolar rhabdomyosarcoma, bladder cancer (e.g., bladder carcinoma),bone cancer, brain cancer (e.g., medulloblastoma), breast cancer, cancerof the anus, anal canal, or anorectum, cancer of the eye, cancer of theintrahepatic bile duct, cancer of the joints, cancer of the neck,gallbladder, or pleura, cancer of the nose, nasal cavity, or middle ear,cancer of the oral cavity, cancer of the vulva, chronic lymphocyticleukemia, chronic myeloid cancer, colon cancer, esophageal cancer,cervical cancer, fibrosarcoma, gastrointestinal carcinoid tumor, headand neck cancer (e.g., head and neck squamous cell carcinoma), Hodgkinlymphoma, hypopharynx cancer, kidney cancer, larynx cancer, leukemia,liquid tumors, liver cancer, lung cancer (e.g., non-small cell lungcarcinoma), lymphoma, malignant mesothelioma, mastocytoma, melanoma,multiple myeloma, nasopharynx cancer, non-Hodgkin lymphoma, B-chroniclymphocytic leukemia, hairy cell leukemia, acute lymphocytic leukemia(ALL), and Burkitt's lymphoma, ovarian cancer, pancreatic cancer,peritoneum, omentum, and mesentery cancer, pharynx cancer, prostatecancer, rectal cancer, renal cancer, skin cancer, small intestinecancer, soft tissue cancer, solid tumors, stomach cancer, testicularcancer, thyroid cancer, and ureter cancer. Preferably, the cancer is ahematological malignancy (e.g., leukemia or lymphoma, including but notlimited to Hodgkin lymphoma, non-Hodgkin lymphoma, chronic lymphocyticleukemia, acute lymphocytic cancer, acute myeloid leukemia, B-chroniclymphocytic leukemia, hairy cell leukemia, acute lymphocytic leukemia(ALL), and Burkitt's lymphoma). Preferably, the cancer is characterizedby the expression of CD22.

The terms “treat,” and “prevent” as well as words stemming therefrom, asused herein, do not necessarily imply 100% or complete treatment orprevention. Rather, there are varying degrees of treatment or preventionof which one of ordinary skill in the art recognizes as having apotential benefit or therapeutic effect. In this respect, the inventivemethods can provide any amount of any level of treatment or preventionof cancer in a mammal. Furthermore, the treatment or prevention providedby the inventive method can include treatment or prevention of one ormore conditions or symptoms of the disease, e.g., cancer, being treatedor prevented. Also, for purposes herein, “prevention” can encompassdelaying the onset of the disease, or a symptom or condition thereof.

Another embodiment of the invention provides a use of the inventiveCARs, nucleic acids, recombinant expression vectors, host cells,populations of cells, antibodies, or antigen binding portions thereof,or pharmaceutical compositions, for the treatment or prevention ofcancer in a mammal.

Another embodiment of the invention provides a method of detecting thepresence of cancer in a mammal, comprising: (a) contacting a samplecomprising one or more cells from the mammal with the CARs, the nucleicacids, the recombinant expression vectors, the host cells, thepopulation of cells, the antibodies, and/or the antigen binding portionsthereof of the invention, thereby forming a complex, (b) and detectingthe complex, wherein detection of the complex is indicative of thepresence of cancer in the mammal.

The sample may be obtained by any suitable method, e.g., biopsy ornecropsy. A biopsy is the removal of tissue and/or cells from anindividual. Such removal may be to collect tissue and/or cells from theindividual in order to perform experimentation on the removed tissueand/or cells. This experimentation may include experiments to determineif the individual has and/or is suffering from a certain condition ordisease-state. The condition or disease may be, e.g., cancer.

With respect to an embodiment of the inventive method of detecting thepresence of cancer in a mammal, the sample comprising cells of themammal can be a sample comprising whole cells, lysates thereof, or afraction of the whole cell lysates, e.g., a nuclear or cytoplasmicfraction, a whole protein fraction, or a nucleic acid fraction. If thesample comprises whole cells, the cells can be any cells of the mammal,e.g., the cells of any organ or tissue, including blood cells orendothelial cells.

For purposes of the inventive detecting method, the contacting can takeplace in vitro or in vivo with respect to the mammal. Preferably, thecontacting is in vitro.

Also, detection of the complex can occur through any number of waysknown in the art. For instance, the inventive TCRs, polypeptides,proteins, nucleic acids, recombinant expression vectors, host cells,populations of cells, or antibodies, or antigen binding portionsthereof, described herein, can be labeled with a detectable label suchas, for instance, a radioisotope, a fluorophore (e.g., fluoresceinisothiocyanate (FITC), phycoerythrin (PE)), an enzyme (e.g., alkalinephosphatase, horseradish peroxidase), and element particles (e.g., goldparticles).

Methods of testing a CAR for the ability to recognize target cells andfor antigen specificity are known in the art. For instance, Clay et al.,J. Immunol., 163: 507-513 (1999), teaches methods of measuring therelease of cytokines (e.g., interferon-γ, granulocyte/monocyte colonystimulating factor (GM-CSF), tumor necrosis factor a (TNF-α) orinterleukin 2 (IL-2)). In addition, CAR function can be evaluated bymeasurement of cellular cytoxicity, as described in Zhao et al., J.Immunol., 174: 4415-4423 (2005).

The following examples further illustrate the invention but, of course,should not be construed as in any way limiting its scope.

Example 1

This example demonstrates the synthesis of anti-CD22 CARs, transductionof PBMC with anti-CD22 CARs, and analysis of CAR surface expression ontransduced PBMC.

CAR-encoding sequences were synthesized using codon-optimizationalgorithms (Mr. Gene GmBH, Regensburg, Germany) and subcloned into“destination” vectors as described in (Zhao et al., J. Immunol.,183(9):5563-74 (2009)) encoding a first version second generation CAR(CD28 transmembrane and intracellular T cell signaling domains andCD3-zeta chain intracellular T cell signaling domain), second versionsecond generation CAR (CD8 transmembrane domain linked to CD137 andCD3-zeta T cell signaling domains), or a third generation CAR (CD8transmembrane domain linked to CD28, CD137, and CD3-zeta intracellular Tcell signaling domains) sequences as shown in Table A.

TABLE A Antigen Binding Further SEQ ID NO: Domain ComponentsHA22CAR-second HA22 CH2CH3 generation, CD28 transmembrane domain version1 CD28 and CD3ζ intracellular T cell signaling domains HA22 CAR-thirdHA22 CH2CH3 generation CD8 transmembrane domain CD28, CD137, and CD3ζintracellular T cell signaling domains HASH22 CAR-second HA22 shortimmunoglobulin generation, constant domain sequence version 1 CD28transmembrane domain CD28 and CD3ζ intracellular T cell signalingdomains HASH22 CAR-third HA22 short immunoglobulin generation constantdomain sequence CD8 transmembrane domain CD28, CD137, and CD3ζintracellular T cell signaling domains BL22 CAR-second BL22 CH2CH3generation CD28 transmembrane domain CD28 and CD3ζ intracellular T cellsignaling domains BL22 CAR-third BL22 CH2CH3 generation CD8transmembrane domain CD28, CD137, and CD3ζ intracellular T cellsignaling domains HASH22 CAR-second HA22 short immunoglobulingeneration, constant domain sequence version 2 CD8 transmembrane domainCD137 and CD3ζ intracellular T cell signaling domains m971 CAR-secondm971 CD28 transmembrane domain generation, CD28 and CD3ζ intracellularversion 1 T cell signaling domains (SEQ ID NO: 23) m971 CAR-third m971CD8 transmembrane domain generation CD28, CD137, and CD3ζ intracellular(SEQ ID NO: 24) T cell signaling domains m971 CAR-second m971 CD8transmembrane domain generation, CD137 and CD3ζ intracellular version 2T cell signaling domains (SEQ ID NO: 22)

Retroviral vector supernatants were created by transfecting 293GP cellswith plasmids encoding CAR retroviral vectors and the RD114 envelopeglycoprotein, collecting culture supernatants (s/n) 48-72 hours later.The culture supernatants were frozen or used immediately to transduceOKT3 and IL-2 activated human PBMC using the “on plate” method for 2consecutive days (culture of lymphocytes on plates coated withRECTRONECTIN (Takara Bio Inc., Shiga, Japan) pre-exposed to dilutions ofvector containing s/n) as previously described in Y. Zhao et al., J.Immunol., 183: 5563 (2009). Also used in this study was retroviral s/ncontaining a CD19-specific CAR from a permanent producer cell line(Kochenderfer et al., Blood, 116: 4099 (2010)).

CAR expression on transduced T cells was determined by flow cytometry.To directly measure the number of cells able to bind CD22 by virtue ofthe CH2CH3 domain, goat anti-human IgG (H&L) (Invitrogen, Grand Island,N.Y.) was used. To detect non-CH2CH3 encoding CARs, CD22-Fc (R&DSystems) followed by anti IgG-Fc PE (Jackson ImmunoResearch, West Grove,Pa.). The HA22SH CAR expresses a short immunoglobulin constant domainsequence instead of CH2CH3. The CD19-CAR was detected using Protein L.Biotinylated protein L (50 ng/ul, Thermo Scientific, Waltham, Mass.) wasbound, cells washed, then detected with SA-PE (4 ug/ml, BD Biosciences,Franklin Lakes, N.J.). For comparison, a CD19-CAR vector s/n was alsoevaluated. Flow cytometry experiments confirmed CAR expression ontransduced T cells. Second generation CARs demonstrated increasedsurface expression levels as compared to third generation CARs asmeasured by mean fluorescence intensity (MFI).

Example 2

This example demonstrates the expression of CD22 and CD19 antigens onleukemia cell lines.

Human leukemia cell lines (REH, SEM, NALM6, KOPN8, Daudi, Raji, andK562) were evaluated for the expression level of CD19 and CD22 on thecell surface using QUANTI-BRITE PE beads (BD Biosciences) and PE-labeledanti-CD19 and anti-CD22 antibody (Table 2). “Receptor Number Per Cell”indicates the approximate absolute number of molecules per cell on eachof the indicated cell lines. Data were calculated by determiningantibodies bound per cell (ABC) using the CELLQUEST software (BD) dataanalysis tools in accordance with the manufacturer's instructions.

TABLE 2 Leukemia Cell Line Receptor Number Per Cell REH CD19 15,100 SEMCD19 50,800 NALM-6 CD19 50,500 KOPN-8 CD19 60,800 Daudi CD19 15,000 RajiCD19 50,000 K562 CD19 <100 REH CD22 7,000 SEM CD22 7,000 NALM-6 CD228,000 KOPN-8 CD22 15,300 Daudi CD22 8,000 Raji CD22 60,800 K562 CD22<200

Example 3

This example demonstrates the lytic activity of cells expressing thesecond generation (version 1) m971 CAR.

To determine the lytic activity, leukemia cell lines (REH, SEM, NALM6,KOPN8, Daudi, Raji, and K562) were ⁵¹Cr labeled and used as targets incytotoxicity assays. Cells were untransduced (mock) or were transducedwith the second generation (version 1) m971 CAR (SEQ ID NO: 23), theanti-CD19 CAR, or the second generation (version 1) HASH22 CAR and wereused as effector cells at various effector to target ratios in thecytotoxicity assays. Percent lysis of the target cells was measured andis shown in FIG. 1A-1G.

As shown in FIGS. 1A-1G, the cells transduced with the second generation(version 1) m971 CAR (SEQ ID NO: 23) lysed CD22-expressing leukemia celllines REH, SEM, NALM6, KOPN8, Daudi, and Raji, and did not lyse non-CD22expressing cell line K562. Cells transduced with the second generation(version 1) m971 CAR (SEQ ID NO: 23) demonstrated superior lytic abilityas compared to cells transduced with the second generation (version 1)HASH22 CAR.

Seven BCP-ALL patient samples were analyzed for CD22 and CD19expression. A wide range of antigen expression was seen, as demonstratedby flow cytometric analysis. Some patients showed strong positivity forboth antigens while others had decreased CD22 staining. In each case,CD22-specific CARs conferred anti-leukemic lytic activity on third partylymphocytes.

Example 4

This example demonstrates the lytic activity of T cells expressing anm971 CAR.

To determine if second or third generation CAR constructs providedincreased lytic activity, leukemia cell lines Raji (CD22^(hi)), NALM6-GL(CD22^(low)), or K562 (CD22-negative) were ⁵¹Cr labeled and used astargets in cytotoxicity assays. Effector cells were human T cells thatwere untransduced (mock) or transduced with m971-second generation,version 1 CAR (SEQ ID NO: 23) or m971-third generation CAR (SEQ ID NO:24). Effector cells were co-cultured with target cells at variouseffector to target (E:T) ratios. The results are shown in FIGS. 2A-2C.

As shown in FIGS. 2A-2C, second generation m971 CARs demonstratedsuperior lytic activity as compared to third generation m971 CARs.

Example 5

This example demonstrates the reactivity of T cells expressing an m971CAR.

T cells that were untransduced (mock) or transduced with one of thefollowing CARs: anti-CD19, m971-second generation version 1 (SEQ ID NO:23), m971-third generation (SEQ ID NO: 24), HASH22-second generationversion 1, HASH22-second generation version 2, or HASH22-thirdgeneration were co-cultured with leukemia cell lines Raji (CD22^(hi)),NALM6-GL (CD22^(low)), or K562 (CD22-negative), and the amounts ofinterferon (IFN)-γ, tumor necrosis factor (TNF) a, and interleukin(IL)-2 secreted were measured. The results are shown in FIGS. 3A-3I.

As shown in FIGS. 3A-3I, cells transduced with m971-second generation,version 1 CAR secreted higher amounts of IFN-γ, IL-2, and TNFα inresponse to co-culture with CD22 expressing cell lines as compared tothird generation m971 CARs.

Example 6

This example demonstrates that cells transduced with the m971 CARretards the progression of disease and lengthens the duration ofsurvival in vivo as compared to an HA22 CAR.

NOD scid gamma, immune-deficient (NSG) mice were injected on day 0 with0.5×10⁶ NALM6-GL (NAML6 transfected with Luciferase). On Day 3, micewere treated with 1×10⁷ untransduced (mock) T cells or T cellstransduced with HASH22-second generation, version 1 CAR or m971-secondgeneration, version 1 CAR (SEQ ID NO: 23). Tumor burden was measured interms of bioluminescent signals per each mouse as photons/s/cm²/sr withbioluminescent imaging using the Xenogen IVIS instrument. Mice wereinjected intraperitoneally (i.p.) with 3 mg D-luciferin (Caliper LifeSciences, Inc., Hopkinton, Mass.) and 4 minutes post-injection,anesthetized mice were imaged with an exposure time of 30 seconds.LIVING IMAGE software was used to analyze the bioluminescent signals pereach mouse as photons/s/cm²/sr on days 3, 7, and 15. Signal intensitywas plotted over time, and the results are shown in FIG. 4A.

As shown in FIG. 4A, all mice had equivalent disease on Day 3. Micetreated with T cells transduced with m971-second generation, version 1CAR (SEQ ID NO: 23) reduced the tumor burden in mice as compared to micetreated with control cells or cells transduced with HA22 CAR.

Survival of the mice was measured for 50 days, survival statistics werecalculated using Log-rank (Mantel-Cox) analysis, and the survivalresults are shown in FIG. 4B. As shown in FIG. 4B, mice treated with Tcells transduced with m971-second generation, version 1 CAR (SEQ ID NO:23) demonstrated increased survival as compared to mice treated withcontrol cells transduced with HA22 CAR.

To determine if treatment failure was due to antigenic loss, mice wereeuthanized due to disease burden according to institutional protocol,and spleens analyzed by flow cytometry. Prior to therapy, high levels ofCAR expression were seen on HASH-28z and m971-28z transduced T cells,and the NALM6-GL cells expressed both CD19 and CD22. Upon sacrifice,spleens were analyzed first for human CD45 and GFP expression. Gated GFPpositive cells represent tumor, and upon further analysis this gatepopulation continued to express GFP. However, any CD45 positive and GFPnegative cells remaining no longer expressed the anti-CD22 CAR. Thisindicates that the leukemia continued to express CD22 and that CAR Tcells did not persist at the time of sacrifice. When the experiment wasrepeated and mice sacrificed on day 12, when therapeutic T cells werestill having an effect, they could readily be detected in the blood,spleen, and bone marrow. The m971-28z (second generation, version 1) CARshowed a much greater ability to infiltrate the bone marrow.

Example 7

This example demonstrates a dose response curve for the adoptivetransfer of cells expressing an m971 CAR.

NSG (immunodeficient) mice were injected with 0.5×10⁶ NALM6-GL cells onday 0. On day 3, the animals were imaged using luciferin substrate toconfirm the presence of leukemia. On day 3, leukemia-bearing mice wereinjected intravenously (i.v.) with untransduced (mock) T cells or15×10⁶, 5×10⁶, 1×10⁶, or 1×10⁵ T cells transduced with a nucleotidesequence encoding a m971-second generation, version 2 CAR (SEQ ID NO:22). The CAR transduction efficiency of the transduced T-cells was 90%.T cells were stimulated with anti-CD3/CD28 beads (1:1), cultured in 40units/ml interleukin-2, and injected into the mice 10 days after theinitial stimulation. Mice were imaged again on day 5 and day 8. Theresults are shown in FIGS. 5 and 6. A change in shading from grey towhite indicates increased tumor burden in FIG. 5. In FIG. 6, a decreasein photons indicates a decrease in tumor burden. As shown in FIGS. 5 and6, complete elimination of the leukemia was seen with the highest doselevel by day 8.

All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference to the sameextent as if each reference were individually and specifically indicatedto be incorporated by reference and were set forth in its entiretyherein.

The use of the terms “a” and “an” and “the” and “at least one” andsimilar referents in the context of describing the invention (especiallyin the context of the following claims) are to be construed to coverboth the singular and the plural, unless otherwise indicated herein orclearly contradicted by context. The use of the term “at least one”followed by a list of one or more items (for example, “at least one of Aand B”) is to be construed to mean one item selected from the listeditems (A or B) or any combination of two or more of the listed items (Aand B), unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising,” “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted. Recitation of ranges of valuesherein are merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range, unlessotherwise indicated herein, and each separate value is incorporated intothe specification as if it were individually recited herein. All methodsdescribed herein can be performed in any suitable order unless otherwiseindicated herein or otherwise clearly contradicted by context. The useof any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise claimed. No language in the specification should be construedas indicating any non-claimed element as essential to the practice ofthe invention.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention.Variations of those preferred embodiments may become apparent to thoseof ordinary skill in the art upon reading the foregoing description. Theinventors expect skilled artisans to employ such variations asappropriate, and the inventors intend for the invention to be practicedotherwise than as specifically described herein. Accordingly, thisinvention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

1.-23. (canceled)
 24. A method of treating a cancer that expresses CD22in a mammal, the method comprising administering to the mammal aneffective amount of a chimeric receptor (CAR) to treat the cancer thatexpresses CD22 in the mammal; wherein the CAR comprises an antigenbinding domain comprising the amino acid sequence of SEQ ID NOs: 1-6, atransmembrane domain, and an intracellular T cell signaling domain. 25.The method of claim 24, wherein the cancer that expresses CD22 is aleukemia or a lymphoma.
 26. The method of claim 25, wherein the leukemiais chronic lymphocytic leukemia, acute myeloid leukemia, hairy cellleukemia, or acute lymphocytic leukemia (ALL).
 27. The method of claim26, wherein the leukemia is acute lymphocytic leukemia (ALL).
 28. Themethod of claim 26, wherein the leukemia is hairy cell leukemia.
 29. Themethod of claim 25, wherein the lymphoma is Hodgkin lymphoma,non-Hodgkin lymphoma or Burkitt's lymphoma.
 30. The method according toclaim 24, wherein the antigen binding domain comprises a light chainvariable region comprising an amino acid sequence comprising SEQ ID NO:7.
 31. The method according to claim 24 or 30, wherein the antigenbinding domain comprises a heavy chain variable region comprising anamino acid sequence comprising SEQ ID NO:
 8. 32. The method according toclaim 24, wherein the antigen binding domain comprises an amino acidsequence comprising SEQ ID NO:
 9. 33. The method according to claim 24,wherein the antigen binding domain comprises a linker comprising SEQ IDNO:
 11. 34. The method according to claim 24, further comprising aleader sequence comprising SEQ ID NO:
 10. 35. The method according toclaim 24, wherein the transmembrane domain comprises i) a transmembraneportion of a CD8 and/or ii) a transmembrane portion of a CD28.
 36. Themethod according to claim 24, wherein the transmembrane domain comprisesan amino acid sequence comprising SEQ ID NO: 12 or 18 and/or an aminoacid sequence comprising SEQ ID NO:
 15. 37. The method according toclaim 24, wherein the intracellular T cell signaling domain comprisesone or more of i) an intracellular signaling portion of a CD28, ii) anintracellular signaling portion of CD137, and iii) an intracellularsignaling portion of a CD3 zeta.
 38. The method according to claim 24,wherein the intracellular T cell signaling domain comprises an aminoacid sequence comprising SEQ ID NO: 16 or
 19. 39. The method accordingto claim 24, wherein the intracellular T cell signaling domain comprisesan amino acid sequence comprising SEQ ID NO: 13 or
 20. 40. The methodaccording to claim 24, wherein the intracellular T cell signaling domaincomprises an amino acid sequence comprising SEQ ID NO: 14, 17 or
 21. 41.The method according to claim 24, comprising an amino acid sequencecomprising any one of SEQ ID NOs: 22-24.
 42. The method according toclaim 24, wherein the intracellular T cell signaling domain comprises anintracellular portion of a CD28 or comprises an intracellular signalingportion of a CD137 and wherein the CAR does not comprise anintracellular signaling domain of both CD28 and CD137.
 43. The method ofclaim 42, wherein the CAR does not comprise an intracellular signalingdomain derived from more than one T cell costimulatory molecule.
 44. Themethod of claim 24, wherein the CAR comprises an amino acid sequencecomprising any one of SEQ ID NOs: 22-24.